Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis

ABSTRACTDiagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs andLeishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi,Leishmania braziliensis,Ehrlichia canis, andBabesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected withB. canisandE. canis(100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.

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Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

Clinical and Vaccine Immunology ◽

10.1128/cvi.00583-13 ◽

2013 ◽

Vol 21 (1) ◽

pp. 96-106 ◽

Cited By ~ 18

Author(s):

Lourena E. Costa ◽

Mayara I. S. Lima ◽

Miguel A. Chávez-Fumagalli ◽

Daniel Menezes-Souza ◽

Vivian T. Martins ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Phage Display ◽

Leishmania Infantum ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Phage Display Technology ◽

Predictive Values ◽

Content Type ◽

Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

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Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs

European Journal of Microbiology and Immunology ◽

10.1556/1886.2020.00018 ◽

2020 ◽

Vol 10 (3) ◽

pp. 165-171

Author(s):

Ingrid E. Pereira ◽

Kyssia P. Silva ◽

Laura M. Menegati ◽

Aimara C. Pinheiro ◽

Elaine A. O. Assunção ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Recombinant Proteins ◽

Serological Test ◽

High Sensitivity ◽

High Specificity ◽

Cross Reactivity ◽

Reservoir Host ◽

Superior Performance ◽

Canine Visceral Leishmaniasis ◽

Roc Curve Analysis

AbstractControl of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

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Serological reactivity of different antigenic preparations of Leishmania (Leishmania) amazonensis and the Leishmania braziliensis complex

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822008000200001 ◽

2008 ◽

Vol 41 (2) ◽

pp. 135-141 ◽

Cited By ~ 11

Author(s):

Adriano Gomes-Silva ◽

Maria Aparecida Souza ◽

Sandra Regina Afonso-Cardoso ◽

Lívia Resende Andrade ◽

Reynaldo Dietze ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Affinity Chromatography ◽

Healthy Volunteers ◽

Concanavalin A ◽

Leishmania Amazonensis ◽

Leishmania Braziliensis ◽

Elisa Assay ◽

Serum Samples ◽

Sensitivity Range ◽

Serological Reactivity

Total antigen from Leishmania (Leishmania) amazonensis and isolates from the Leishmania braziliensis complex, along with their respective antigenic fractions obtained by affinity chromatography on concanavalin-A-Sepharose and jacalin-agarose columns evaluated using immunoenzymatic ELISA assay. For this, serum samples from 229 patients were used, grouped as American tegmental leishmaniasis (nº=58), visceral leishmaniasis (nº=28), Chagas disease (nº=49), malaria (nº=32), tuberculosis (nº=13) and healthy volunteers (nº=49). Samples from American tegmentary leishmaniasis showed higher reactivity with antigens isolated from the Leishmania braziliensis complex than with antigens from Leishmania amazonensis (p<0.001). ELISA assays showed a sensitivity range from 60% to 95% with antigens isolated from the Leishmania braziliensis complex. There was marked nonspecific reactivity among serum samples with the use of antigenic fractions binding with concanavalin-A and jacalin from both Leishmania complexes, in comparison with other antigens (p<0.001). The results presented in this study suggest that the use of homologous antigens increases the efficiency of anti-Leishmania immunoglobulin detection, which may be very valuable for diagnostic purposes.

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Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia) braziliensis

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651996000300003 ◽

1996 ◽

Vol 38 (3) ◽

pp. 177-185 ◽

Cited By ~ 51

Author(s):

Ana de Cássia Vexenat ◽

Jaime M. Santana ◽

Antonio R.L. Teixeira

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Cross Reactivity ◽

Antigenic Determinants ◽

Leishmania Braziliensis ◽

Leishmania Chagasi ◽

Mucocutaneous Leishmaniasis ◽

Kala Azar ◽

Homologous Antigen ◽

Positive Results

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

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Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46652011000500008 ◽

2011 ◽

Vol 53 (5) ◽

pp. 283-289 ◽

Cited By ~ 3

Author(s):

Flávia Coelho Ribeiro ◽

Armando de O. Schubach ◽

Eliame Mouta-Confort ◽

Tânia M.V. Pacheco ◽

Maria de Fátima Madeira ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Large Scale ◽

Cross Reactivity ◽

Leishmania Braziliensis ◽

Leishmania Chagasi ◽

Serum Samples ◽

Canine Ehrlichiosis ◽

Control Serum ◽

Homologous Antigen ◽

Heterologous Antigens

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

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Specific Serodiagnosis of Canine Visceral Leishmaniasis Using Leishmania Species Ribosomal Protein Extracts

Clinical and Vaccine Immunology ◽

10.1128/cvi.00295-09 ◽

2009 ◽

Vol 16 (12) ◽

pp. 1774-1780 ◽

Cited By ~ 25

Author(s):

Eduardo A. F. Coelho ◽

Laura Ramírez ◽

Mariana A. F. Costa ◽

Vinicio T. S. Coelho ◽

Vivian T. Martins ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Ribosomal Proteins ◽

High Sensitivity ◽

Leishmania Species ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Canine Visceral Leishmaniasis ◽

Leishmania Chagasi ◽

Serum Samples ◽

Potential Tool

ABSTRACT In the present work, we have analyzed the antigenicity of Leishmania species ribosomal proteins (LRPs). To accomplish this, Leishmania infantum ribosomes were biochemically purified from promastigote cytosolic extracts, and their reactivities were analyzed by using the sera from dogs naturally infected with L. infantum. Since antibodies reacting against different ribosomal proteins were observed in all the serum samples obtained from dogs with symptomatic visceral leishmaniasis tested, we have analyzed the potential usefulness of the LRP extracts in the development of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of canine visceral leishmaniasis (CVL) in an area of Brazil where visceral leishmaniasis is endemic due to infection by Leishmania chagasi. A comparative ELISA with crude soluble Leishmania chagasi antigen (SLA) and L. infantum LRPs was performed. LRP- and SLA-based ELISAs gave similar sensitivities for the diagnosis of symptomatic CVL, but the LRP extract provided a very high sensitivity for the detection of oligosymptomatic and asymptomatic dogs. In addition, an LRP-based ELISA showed a higher specificity when the sera from dogs harboring other infections were included in the analysis. The LRP antigen displayed no cross-reactivity with sera from dogs that had any of the other diseases tested, notably, Chagas' disease. Our findings suggest that LRPs are a potential tool for the diagnosis of CVL and will be particularly useful for the diagnosis of asymptomatic CVL.

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Development of an Immunochromatographic Assay Kit Using Fluorescent Silica Nanoparticles for Rapid Diagnosis of Acanthamoeba Keratitis

Journal of Clinical Microbiology ◽

10.1128/jcm.02595-14 ◽

2014 ◽

Vol 53 (1) ◽

pp. 273-277 ◽

Cited By ~ 10

Author(s):

Koji Toriyama ◽

Takashi Suzuki ◽

Tomoyuki Inoue ◽

Hiroshi Eguchi ◽

Saichi Hoshi ◽

...

Keyword(s):

Silica Nanoparticles ◽

Real Time ◽

Real Time Pcr ◽

High Sensitivity ◽

Acanthamoeba Keratitis ◽

Cross Reactivity ◽

Immunochromatographic Assay ◽

Content Type ◽

Fluorescent Silica Nanoparticles ◽

Positive Results

We developed an immunochromatographic assay kit that uses fluorescent silica nanoparticles bound to anti-Acanthamoebaantibodies (fluorescent immunochromatographic assay [FICGA]) and evaluated its efficacy for the detection ofAcanthamoebaand diagnosis ofAcanthamoebakeratitis (AK). The sensitivity of the FICGA kit was evaluated using samples ofAcanthamoebatrophozoites and cysts diluted to various concentrations. A conventional immunochromatographic assay kit with latex labels (LICGA) was also evaluated to determine its sensitivity in detectingAcanthamoebatrophozoites. To check for cross-reactivity, the FICGA was performed by using samples of other common causative pathogens of infectious keratitis, such asPseudomonas aeruginosa,Staphylococcus aureus,Staphylococcus epidermidis, andCandida albicans. Corneal scrapings from patients with suspected AK were tested with the FICGA kit to detect the presence ofAcanthamoeba, and the results were compared with those of real-time PCR. The FICGA kit detected organisms at concentrations as low as 5 trophozoites or 40 cysts per sample. There were no cross-reactivities with other pathogens. The FICGA was approximately 20 times more sensitive than the LICGA for the detection ofAcanthamoebatrophozoites. The FICGA kit yielded positive results for all 10 patients, which corresponded well with the real-time PCR results. The FICGA kit demonstrated high sensitivity for the detection ofAcanthamoebaand may be useful for the diagnosis of AK.

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Cross-Reactivity Using Chimeric Trypanosoma cruzi Antigens: Diagnostic Performance in Settings Where Chagas Disease and American Cutaneous or Visceral Leishmaniasis Are Coendemic

Journal of Clinical Microbiology ◽

10.1128/jcm.00762-19 ◽

2019 ◽

Vol 57 (8) ◽

Cited By ~ 2

Author(s):

Ramona Tavares Daltro ◽

Leonardo Maia Leony ◽

Natália Erdens Maron Freitas ◽

Ângelo Antônio Oliveira Silva ◽

Emily Ferreira Santos ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Chagas Disease ◽

Diagnostic Performance ◽

Latent Class ◽

Cross Reactivity ◽

Chimeric Proteins ◽

Serum Samples ◽

Perfect Agreement ◽

Content Type ◽

Chronic Chagas Disease

ABSTRACT Chimeric T. cruzi antigens have been proposed as a diagnostic tool for chronic Chagas disease (CD) in both settings where Chagas disease is endemic and those where it is not endemic. Antibody response varies in accordance to each T. cruzi strain, presenting challenges to the use of antigens lacking demonstrated cross-reactivity with Leishmania spp. Our group expressed four chimeric proteins (IBMP-8.1, IBMP-8.2, IBMP-8.3, and IBMP-8.4) and previously assessed their diagnostic performance to determine cross-reactivity with Leishmania spp. Here, we validated our findings using serum samples from different Brazilian geographic areas reporting endemic Chagas disease, endemic visceral or American cutaneous leishmaniasis (ACL), or both. Overall, 829 serum samples were evaluated using commercial and IBMP enzyme-linked immunosorbent assays. Due to the absence of a reference assay to diagnosis CD, latent class analysis (LCA) was performed through the use of a statistical model. The incidence of cross-reactivity for ACL-positive samples varied from 0.35% (IBMP-8.3) to 0.70% (IBMP-8.1 and IBMP-8.2). Regarding visceral leishmaniasis (VL)-positive samples, the IBMP-8.2 and IBMP-8.3 antigens cross-reacted with six (3.49%) and with only one sample (0.58%), respectively. No cross-reactivity with either ACL or VL was observed for the IBMP-8.4 antigen. Similarly, no cross-reactions were found when VL-positive samples were assayed with IBMP-8.1. The agreement among the results obtained using IBMP antigens ranged from 97.3% for IBMP-8.2 and 99% for IBMP-8.1 and IBMP-8.3 to 100% for IBMP-8.4, demonstrating almost perfect agreement with LCA. Accordingly, in light of the negligible cross-reactivity with both ACL and VL, we suggest the use of IBMP antigens in regions where T. cruzi and Leishmania spp. are coendemic.

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